ProjectSummary Whilecheckpointblockadehasprovidedunprecedentedclinicalbenefit,therearestillmanycancerpatientswho remain unresponsive to therapy. Given that the presence of tumor-infiltrating CD8+ T cells predicts clinical responses, there has been increased focus on what spontaneously generates this ?inflamed? tumor microenvironment.Inansweringthisquestion,currentapproacheshaveidentifiedphagocytosedtumorDNAas the ligand that activates cGAS-STING pathway in BATF3-dependent DCs to elicit type I IFN signaling that ultimatelymaturesDCsandeffectivelyprimesTcells.Theseapproaches,however,dependprimarilyontumor models that highly express infectious endogenous retroviruses (ERVs), which is absent in human cancers. Becausethere is the possibility that type I IFN and innate sensorsarebeing erroneously induced,wepropose touseERV-freeimmunogenicYUMMERmelanomalinesdevelopedbyMarcusBosenbergtostudytheinnate sensors involved in activating DCs. Based on preliminary data demonstrating that tumor rejection is STING- dependentbutcGAS-independentandrecentstudiesshowingthatchromosomalinstability(CIN)incancercan autonomouslyactivatecGAS-STING-NFkBpathway,wehypothesizethatCINisactivatingtumorproductionof cGAMPwhichthentransactivatesSTINGinnearbyBATF3-dependentDCs,leadingtoNFkBsignalingandDC maturation.Todirectlytestthis,Iwillpursuethefollowingaims.Formyfirstaim,Iwilldeterminethehostcell type in which STING expression is required for cancer immunosurveillance. To accomplish this, I will subcutaneouslyimplantYUMMERcellsinmicelackingvariousDClineagesandscreenforanydeficienciesin tumorrejection,trackingtumorvolumeandmeasuringDCactivationandTcellinfiltrationbyflowcytometry.For mysecondaim,Iwillidentifythetumor-derivedligandthatisresponsibleforactivatinghostSTING.To do this, I will induce CIN in YUMM cells (the non-immunogenic parental cell line of YUMMER lines) by overexpressingthedominantnegativealleleofmicrotubuledepolymerizingkinesin(MCAK)totestwhetherCIN issufficienttoinduceahostSTING-dependentrejection.WewillalsostablyknockoutcGASinYUMMERcells toassesswhetherspontaneousrejectioncanbeabrogatedintheabsenceoftumorcGAMPproduction.Formy third aim, I will elucidate the downstream signaling pathway elicited by host STING. By conditionally knockingoutNFkBpathwaycomponentsintheidentifiedDClineage,wewillverifywhetherNFkBisrequiredfor cancer immunosurveillance. Next, we will utilize RNA-Seq of sorted DCs to identify potential cytokines driving tumorrejectionandverifythattheseidentifiedcytokinesarerequiredforimmunosurveillancebyadministration of depleting cytokines. If our hypothesis proves true, we will have identified how the innate immune system broadly recognizes cancer as a pathogen to initiate a specific adaptive response. We expect that our findings willuncovernewstrategiestoconvert?cold?tumorsinto?hot?onesthatcanbetargetedwithcheckpointinhibition.